Monoclonal human natural antibodies

ABSTRACT

Disclosed herein are hydridoma cell lines producing monoclonal human natural IgM antibodies and methods of use thereof. The antibodies are the monoclonal equivalents of circulating human natural antibodies. Also disclosed herein are pharmaceutical formulations and methods for treating HIV-1 infected individuals using the monoclonal human natural antibodies.

This application is a 371 national stage application of PCT/US98/25258,filed Nov. 4, 1998, which claims the benefit of priority to U.S.Provisional Application No. 60/066,463, filed Nov. 24, 1997.

BACKGROUND OF THE INVENTION

The effector molecules of the immune system include a repertoire ofcirculating immunoglobulins non-attributable to exogenous antigenicinduction, variously referred to as “autoantibodies” or “naturalantibodies”. The existence of such antibodies has been long recognizedand their various proposed functions may be classed as “self-attack” or“self-benefit”. For the former, the specter of autoimmunity is raisedand the term “autoantibodies” is customarily applied. For the latter,the term “autoantibodies” is customarily applied. For the latter,designated “natural; antibodies”, support of homeostasis is implied.

U.S. patent application Ser. No. 08/271,210 filed Jul. 5, 1994,discloses a circulating natural human antibody immunoreactive with anarginine-rich epitope present on human protamine. U.S. Pat. No.5,606,026 issued Feb. 25, 1997, discloses that the arginine-rich epitopeis present in the Tat protein of HIV-1 and further discloses a secondcirculating human natural antibody immnunoreactive with a differentepitope on the Tat protein of HIV-1. In addition, a third circulatinghuman natural antibody immunoreactive with a cryptic epitope present onhuman lactoferrin is disclosed therein.

It has been shown that all three of the above-mentioned circulatinghuman natural antibodies decrease after HIV infection reaching minimallevels as the patient progresses to AIDS. These antibodies are found inall sera of normal humans of all ages, from cord blood to adult, which,by virtue of their ubiquitous occurrence, are identified as naturalantibodies.

Therefore, what is needed in the art are the monoclonal counterparts ofthese circulating human natural antibodies for their therapeutic anddiagnostic uses.

SUMMARY OF THE INVENTION

The present invention provides monoclonal forms of human naturalantibodies.

In one aspect, the present invention provides hybridoma cell line RWL-1(ATCC CRL 12431), a product of the fusion of Epstein Barr virus (EBV)transformed umbilical cord blood cells and HMMA, mouse: humanheteromyeloma cells.

In another aspect, the present invention provides monoclonal human IgMantibodies, produced by RWL-1 cells.

In yet another aspect, the present invention provides another hybridomacell line, RWT-4 (ATCC CRL 12472), a product of the fusion ofEBV-transformed umbilical cord cells with SHM-D33 cells (ATCC CRL 1668),mouse: human heteromyeloma cells.

In yet another aspect, the present invention provides monoclonal humanIgM antibodies produced by RWT-4 cells.

In a still further aspect, the present invention provides hybridoma cellline RWT-12 (ATCC CRL 12477), a product of the fusion of EBV-transformedhuman umbilical cord cells and HMMA, mouse: human heteromyleoma cells.

In a still further aspect, the present invention provides monoclonalhuman IgM antibodies produced by RWT-4 cells.

In a still further aspect, the present invention provides a method fortreating a patient suffering from an infection caused by HIV-1comprising administering to a patient in need of such treatment aneffective amount for treating said infection of a monoclonal antibodyselected from the group consisting of antibodies produced by RWT-4cells, RWT-12 cells, and mixtures thereof.

In a still further aspect, the present invention provides a method forincreasing CD4+T cells in a patient suffering from an infection causedby HIV-1 comprising administering an amount for increasing CD4+T cellsof antibodies produced by hybridoma cells having Accession Nos. ATCC CRL12472 , ATCC CRL 12477 and mixtures thereof.

In a still further embodiment, the present invention provides apharmaceutical formulation comprising isolated human IgM monoclonalantibodies selected from the group consisting of antibodies produced byhybridoma cell lines having Accession Nos. ATCC CRL 12472, ATCC CRL12477, mixtures thereof and a pharmaceutical acceptable vehicle.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in light of the present description,claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-C) is an SDS PAGE of cyanogen bromide [CNBr] cleavedlactoferrin (LF) and SP80.

A. Protein stain. 1 mol. wt. markers; 2 LF(M); 3 SP80-basic; 4SP80-acidic. All three proteins (2,3,4) show identical cleavagefractions 1-8.

B. Immunotransfer with serum of rabbit immunized with SP80 (acidic andbasic) showing multiplicity of reactive sites and homology of reactivityof LF(M) and SP80.

C. Immunotransfer with normal human male serum showing reactivity solelywith fraction 7 of each of the 3 proteins.

FIG. 2 (A-C) is an Tricine SDS Page.

A. Protein stain. 1 mol. wt. markers; 2 LF(M); 3 SP80 (acidic andbasic). Resolution of fraction 7 shows 2 distinct bands.

B. Immunotransfer with normal human male serum showing reactivityspecifically localized in fraction 7B.

C. Immunoreactivity with fraction 7B of a monoclonal antibody (Mab) IgMfrom a human B cell derived hybridoma.

FIG. 3 (A and B) shows in situ immunoreactivity, displayed by FITClabeled anti-25 human IgM, of a component of human sperm heads with: (A)human serum; (B) Mab reactive with LF fraction 7B.

FIG. 4 (A-C) shows reactivity, by ELISA, of serum (diluted 1:100) ofeach of 5 males, 5 females and the Mab with: (A) 10 μg/ml of thecomplement of sperm coat proteins released following induction of theacrosome reaction in a suspension of swim-up spermatozoa; (B) 10 μg/mlof purified fraction 7B LF(M); C. 10 μg/ml native (non-denatured) LF(M).The relative reactivities of A. and B. indicate that a serum antibodyand the Mab are reactive with a specific component, but not all, of thesperm coat complement. The lack of reactivity with native LF (C)verifies that the natural antibody of serum and the Mab are reactivewith a site of LF that is not revealed in its native state.

FIG. 5. Sequences of the 12—amino acid peptides representing the Tatprotein of HIV-1. Peptides 1-7 (SEQ ID NOS: 1 to 7) and 9-12 (SEQ IDNOS: 9 to 12) represent 5 residue overlaps. Peptide 8 (SEQ ID NO: 8) wasincluded to provide another variant of arginine distribution in order toascertain the maximum reactivity of human sera attributable to thearginine-rich region of Tat. Maximum titer with the arginine-rich region(peptides 7,8,9, SEQ ID NOS: 7, 8, 9) was, in fact, displayed withpeptide 8 (SEQ ID NO: 8). Maximum titer with the cysteine-rich region(peptides 4,5, SEQ ID NOS: 4, 5) was displayed with peptide 4 (SEQ ID NO4).

FIG. 6 (A and B).

A. IgM

B. IgG Analysis of reactivity of two cohorts of 70 human sera, HIV+ andHIV−(normal), with Tat protein of HIV. The HIV+ cohort was assembledfrom sera collected prior to 1994, therefore the characteristics are notattributable to the anti-HIV medications in use since that time. Eachassay plate included both HIV+ and HIV− specimens and a single normalserum (ST). The recorded titer for each serum (X) represents X/ST.

The titers are grouped in intervals of 10 with the number of sera ofeach cohort designated for each interval. The distributions of both IgMand IgG titers for the HIV+ sera are skewed to the lower intervals,particularly those of the IgM.

FIG. 7 (A and B). Distribution of titers of A.IGM and B.IgG, reactivewith peptide 8 (SEQ ID NO: 8) (FIG. 5) in each of two cohorts of 70human sera, HIV+and HIV−(normal). The preponderance of low ,or no titersof IgM and, even more strikingly, of IgG in the HIV+ sera indicates thatdepletion of the natural antibody reactive with the arginine-richsequence of Tat is a correlation of the pathoprogression of HIV.

FIG. 8 (A and B). The distribution of titers of A. IgM and B. IgGreactivity with peptide 4 (SEQ ID NO: 4) (FIG. 5) in two cohorts of 70human sera, HIV+ and HIV−(normal), is in accord with the general trendof lower titers, in HIV+ sera, of the total Tat-reactive antibodies, butless stringent than that demonstrated for the titers of the peptide 8(SEQ ID NO: 8) reactive antibodies (FIG. 11).

FIG. 9 (A and B).

A. CD4+T cell counts.

B. Titers of IgM reactivity with Tat protein, peptide 4 (SEQ ID NO:4)and peptide 8 (SEQ ID NO: 8) (FIG. 5) of serial specimens from an HIV+male over a period of five years preceding his death with a diagnosis ofAIDS. Each specimen for CD4+T cell count was obtained at the same timeas that for serum analysis. The correlation of drop in CD4+T cells withthe decline of titers of the natural antibodies is particularly markedwith respect to the titer of peptide 8 (SEQ ID NO: 8) reactiveantibodies, supporting the proposition that the decline in that naturalantibody may allow the T cell apoptosis, attributed to Tat, to proceed.

FIG. 10 (A and B).

A. CD4+T cell count.

B. Titers of IgM antibodies reactive with Tat protein, peptide 4 (SEQ IDNO:4) and peptide 8 (SEQ ID NO: 8) (FIG. 5) in serial specimens of sera,collected over a period of 9 years, from an HIV+ male whose duration ofinfection is estimated at over 11 years, but has displayed no HIVassociated pathology and has had no anti-HIV medication. Each specimenfor serum analysis was obtained at the same time as that for CD4+cellcount. The titers and pattern of maintenance of titers of the naturalantibodies are correlative with maintenance of the CD4+T cell countswithin the normal range.

FIG. 11 (A and B).

A. CD4+T cell count.

B. Titers of IgM reactive with Tat protein, peptide 4 (SEQ ID NO: 4) andpeptide 8 (SEQ ID NO: 8) (FIG. 5) of serial specimens from an HIV+ male.Following the report of specimen 4, in which decline of CD4+T cell countwas noted, anti-HIV therapy was initiated. The count in specimen 5,taken after 6 months of therapy showed significant rise and the titer ofIgM reactive with peptide 8, (SEQ ID NO: 8) underwent an exceedinglyhigh rise. The successive specimens then showed maintenance of CD4 Tcell counts and natural antibody titers, concomitant with generally goodclinical status.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications and literature references cited hereinare hereby incorporated by reference in their entirety.

In one preferred embodiment, the present invention provides a monoclonalform of a human, natural IgM antibody immunoreactive with a cryticepitope present on human lactoferrin. This antibody is produced byhybridoma RWL-1, deposited with the American Type Culture collection(Manassas, Va.) on Nov. 14, 1997 and received ATCC Accession No. ATCCCRL-12431. The hybridoma was produced by fusing an Epstein Barr virustransformed human umbilical cord cell with a mouse: human heteromyelomacell as described in Example 1 below. The hybridoma produces humanmonoclonal antibodies of the IgM isotype. The fact that the antibodyproducing cell (the human umbilical cord cell) is of neonate origin andthe antibody is of the IgM isotype (and therefore does not cross theplacenta) demonstrates that this is indeed a natural antibody.

The IgM antibody immunoreactive with lactoferrin is characterized as anatural antibody identified since it has been shown to be present in alarge cohort of normal human sera, and for which no pathologic role orassociation is apparent. The reactive site for this natural antibody hasbeen shown previously (3) and confirmed here, to be present in theplasma membrane complex of the human sperm head. These studies, designedto establish the molecular identity of that reactive site, haveconfirmed that an approximately 72.6 kD protein present in seminalplasma (2), accurately determined here as 80 kD, is also present in theprotein coat of the sperm head and that 80 kD protein is homologouswith, and in fact is, lactoferrin. It is shown herein that the notednatural antibody is specifically reactive with LF in a configurationother than that of the LF ubiquitous in body fluids. That configurationand the natural antibody reactivity is revealed, in vitro (FIGS. 2, 4),following denaturation of native circulating LF, and is revealed, invivo (FIG. 3) in the LF incorporated in the protein coat of the humansperm head. LF is present in seminal plasma in the native configurationand, by a mechanism not yet determined, the antibody recognition form isassumed when it is deposited in the spermatozoal membrane/coat complex.The transition to that form and deposition in the sperm surface coatpresumably take place during the period of spermatogenic maturation inthe seminiferous tubules of the testes. It is relevant, therefore, tonote that large molecules such as inununoglobulins, particularly IgM,are excluded from the lumina of the seminiferous tubules (24) and,therefore, from immunoreactivity with sperm components duringspermiogenesis. That barrier, however, does not exist in the femalereproductive tract, where the full complement of circulating antibodiesis present (23). Therefore, the LF reactive natural antibody isavailable for immunoreactivity with the LF of the sperm coat, followingejaculation into the female reproductive tract. That interaction maytake place in the sperm coat in situ as shown (FIG. 3) and is definitelycapable of taking place with the LF released, along with other coat andplasma membrane components (FIG. 4) as the sperm undergoes the sequenceof capacitation and acrosome reaction, which facilitate passage of thesperm through the protective zona pellucida surrounding the oocyte, andsubsequent entry into the oocyte (9, 10). Since the acrosome reactioninvolves fusion of the acrosomal membrane with the plasma membrane, thecomponents of the overlying protein coat are dispersed. Thus, thereleased LF could have ready access to the ooplasm were it not for thepresence, in the fertilization milieu, of the natural antibody capableof immunological nullification of the ability of that LF to endocytosethrough the oocyte membrane and, subsequently, to interact with the DNAof the gametes or pronuclei.

Among the many functions and interactions defined for LF, its capacityto be endocytosed and interact with DNA is of increasing interest (7, 8,27-29). Particularly interesting are the recent reports that theinteraction of LF with DNA is marked by sequence specificity (7). Theunderlying molecular bases for that specificity have not been defined,but it is reasonable to expect that if LF/DNA interaction occurs, invivo it does so within a defmed control system. It is logical, also, topropose that such a system exists in the organized chromosomalcomplement of somatic cells, but not in the nascent undifferentiatedcomplements of the pronuclei. Thus, in that context, the postulatedcontrol of sequence specificity in interaction of LF with DNA may not beoperative. The presence of a natural antibody selectively reactive withLF in the specific configuration in which it exists in the sperm coat,but not with LF in its ubiquitous circulating form, may represent afortuitous natural selection mechanism on two bases: (1) inhibition ofLF interaction with the DNA complements of the fertilized oocyte and (2)restriction of immunoreaction by the circulating natural antibody withLF at other loci, in its more prevalent, important function-servingforms. The innate occurrence of that natural antibody is verified sincethe hybridoma secreting the Mab, utilized to provide significant data ofthis study, was derived from a human cord blood B cell.

As shown below in Example 2, the antibody is immunoreactive with anepitope present on human lactoferrin, specifically the form oflactoferrin present in the protein coat of the human sperm head.Lactoferrin is an 80 kD glycoprotein present in the sperm head.Following induction of the acrosome reaction occurring duringfertilization, lactoferrin (which has been shown to interact with andbind to DNA) could potentially interfere with the interaction of spermand oocyte DNA. Therefore, one of the uses of the antibody of thepresent invention is as an additive to in vitro fertilization reactionsin order to prevent lactoferrin from interacting with sperm DNA prior tofertilization.

In alternative, preferred embodiments of the present invention,hybridoma cells producing monoclonal IgM antibodies immunoreactive withthe Tat protein of HIV-1 are provided. The hybridoma cell lines RWT-4and RWT-12 are immunoreactive with peptide 4 (SEQ ID NO: 4) and peptide8 (SEQ ID NO: 8), respectively, of FIG. 5. These hybridoma cells, as isthe case with hybridoma RWL-1, were produced by fusing EBV-transformedhuman umbilical cord cells with mouse:human heteromyeloma cells. RWT-4cells were deposited with the ATCC on Feb. 12, 1998 and receivedAccession No. ATCC CRL 12472 and RWT-12 cells were deposited on Feb. 25,1998 with the ATCC and received Accession No. ATCC CRL 12477. Theepitope specificity of each antibody is shown below in Example 3.

The monoclonal IgM antibodies produced by hybridomas RWL-1 (ATCC CRL12341) RWT-4 (ATCC CRL 12472) and RWT-12 (ATCC CRL 12477) can beisolated from cultures of the cells that produce them and purified usingtechniques known to those of ordinary skill in the art, such as ammoniumsulfate precipitation, HPLC, column chromatography, etc.

The antibodies of the present invention are the monoclonal equivalentsof the circulating IgM antibody identified in Science 228:1211, 1985(for RWL-1 cells) and described in U.S. Pat. No. 5,606,026 issued Feb.20, 1997 (for RWT-4 and RWT-12 cells). These circulating antibodies aredeficient in HIV-infected individuals and decrease as AIDS approaches.Therefore, the monoclonal antibodies produced by ATCC CRL 12341, ATCCCRL 12477 and ATCC CRL 12472 can be used as positive controls in assaysfor prognosing the onset of AIDS.

In another preferred embodiment of the invention, a method for treatinga patient suffering from an infection caused by HIV-1 comprisingadministering an effective amount to treat HIV-1 of natural human IgMantibodies selected from the group consisting of antibodies produced byRWT-4 cells, antibodies produced by RWT-12 cells and mixtures thereof.It is envisioned that replenishment of the natural antibodies deficientin HIV-1-infected and AIDS patients will be of clinical benefit to theseindividuals.

As shown below in Example 5, the Tat protein of HIV-1 does not stimulatethe induction of antibodies in humans (see Table I of Example 5). Thisobservation coupled with the fact that long term survivors (LTS)/longterm non-progressors (LTNP), patients who are HIV-1 positive but who donot exhibit any symptoms of the disease or progress to AIDS, have normallevels of the circulating natural antibodies equivalent to the IgMantibodies produced by the hybridomas of the present invention. Thisdemonstrates the utility of administering the monoclonal antibodies ofthe present invention as a therapeutic to treat the disease. Due to thefact that the Tat protein of HIV-1 has such an important role inestablishing and maintaining infection, and that the protein does notappear to be immunogenic in humans, administration of the monoclonalantibodies of the present invention to infected individuals is one wayto introduce antibodies specifically directed against the Tat protein.

The data of FIGS. 9, 10 and 11 clearly establish a correspondence, inHIV+ humans, between the CD4+T cell count and the serum titer of the twoIgM natural antibodies reactive with the Tat protein of HIV,specifically with the sequences of the protein represented by peptide 4(SEQ ID NO: 4) and peptide 8 (SEQ ID NO: 8) (FIG. 10). Thecorrespondence is more sharply shown with the antibody reactive withpeptide 8 (SEQ ID NO: 8), representing the arginine-rich sequence ofTat.

Each of FIGS. 9, 10 and 11 display a unique example of thatcorrespondence. FIG. 9 shows the clinical report of CD4+T cell count andthe antibody assay data of a series of corresponding serum specimensfrom an HIV+ male over a period of 5 years preceding his death with adiagnosis of AIDS. FIG. 10 displays the corresponding data of specimensfrom an HIV+ male whose duration of infection is estimated at over 11years, but who has displayed no HIV-associated pathology and has had noanti-HIV medication. FIG. 11 displays the data of specimens from an HIV+male showing that, following institution of anti-HIV medication, bothCD4+T cell count and titers of Tat reactive antibodies, particularly theantibody reactive with peptide 8, rose to levels within the normalrange.

Since various Intravenous IgG (IVIG) preparations currently commerciallyavailable (e.g., from Sandoz Pharmaceuticals or Cutter Biological) havebeen tested and certified for parenteral administration, an IVIGpreparation may be used as a vehicle for administration of themonoclonal IgM antibodies of the present invention. These preparationshave been shown to be safe for human parenteral administration.

Generally, the dosage administered will, of course, vary depending uponknown factors such as age, health and weight of the recipient, type ofconcurrent treatment, frequency of treatment, etc. Usually, a dosage ofactive ingredient can be between about 0.001 and about 10 milligrams perkilogram of body weight. Precise dosage, frequency of administration andtime span of treatment should be monitored, for each individual, bydetermination of rise in CD4+T cell count and other clinical indicia ofrelief from pathogenetic progression.

In yet another preferred embodiment, a method for increasing CD4+T cellcounts in a patient is provided comprising administering to a patient inneed of such treatment, an effective amount to increase CD4+T cellcounts of antibodies selected from the group consisting of antibodiesproduced by hybridoma ATCC CRL 12472, ATCC CRL 12477 and mixturesthereof. The effective amounts are the same as mentioned above.

For parenteral administration, the antibodies of the present inventioncan be formulated into pharmaceutical formulations or dosage forms as asolution, suspension, emulsion, or lyophilied powder in association witha pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose and 5% humanserum albumin. In addition, as mentioned above, IVIG commerciallyavailable preparations can be used as vehicles for delivery of theantibodies of the present invention.

The pharmaceutical formulations of the present inventions do not need toconstitute an effective amount of the antibodies of the presentinventions since such amounts can be achieved by administering aplurality of such formulations.

The present invention is further described below in specific examplewhich are intended to further describe the invention without limitingits scope.

EXAMPLE 1

The hybridoma, RWL-1, which secretes the monoclonal human IgM antibodyreactive with a defined cryptic sequence of human lactoferrin, wascreated by fusion of a human umbilical cord blood B cell with the cellline HMMA, a human/mouse heteromyeloma (Posner MR, Ellorim H, Santos D.(1987) Hybridoma 6:611.) as set forth below.

The cord blood was obtained, at caesarian section, from a normal (butotherwise non-identified) neonate and the mononuclear cells wereisolated by density gradient centrifugation using FICOLL-PAQUE®(available from Pharmacia of Peapack, N.J.) The collected cells werewashed with RPMI-1640 medium (Sigma) and added to a culture ofEpstein-Barr Virus (EBV), (obtained from cultures of ATCC CRL 1612cells) in RPMI 1640 medium and incubated (37° C.) for 2 hours. The cellswere then spun down, resuspended in RPMI 1640, supplemented with fetalcalf serum (FCS), cyclosporin A, Pen/Strep (10 units Penicillin/100 mgStreptomycin per ml), and plated in 96 well plates at 30 cells/well.After 5 weeks incubation at 37° C. with periodic medium replenishment,the culture medium of each well was tested by ELISA (Pruslin FH, WinstonR, Rodman Tc. (1991) J. Immunol. Meth. 137:27) for the IgM monoclonalantibody (Mab) specifically reactive with the 7B fraction of denaturedlactoferrin (FIG. 2C).

Selected cultures of EBV immortalized B cells were grown to a cellconcentration of 10⁶ cells/well, then washed 5× in RPMI-1640(non-supplemented). The fusion partner (HMMA cells, described in Posner,M.R., et al., Hybridoma 6:, 611, 1987) were grown in RPMI 1640, FCS,Pen/Strep and azaguanine, and washed 3× in non-supplemented RPMI-1640°10⁶ cells were mixed with an equal number of the EBV immortalized cells.The mixed cell culture was spun, supernatant decanted and the cellsresuspended in warm (37° C.) 40% polyethylene glycol/RPMI-1640 (pH 7.2)and held for one minute. The cells were spun, washed 2× with RPMI, pH7.8, then resuspended (10⁶ cells/ml) in HY medium (Sigma), supplementedwith 20% FCS, HAT (Sigma), ouabain, Pen/Strep and plated out at 10⁵cells/well. After 3 weeks the growth positive wells were tested for theprevention of the specific antibody. The contents of the wells withpositive antibody were limited out (diluted) and replated at 0.5cells/well in HY/HT (Sigma), and supplemented with 20% FCS, SPIT(Sigma), Pen/Strep. The cells were grown for 5 weeks (37° C.) and thecontents of each well were retested for Mab specificity. Selectedcultures were incubated and grown to density of 10⁶ cells/ml. and spunat 400 RPM. 5 min. Each cell pellet was suspended in 5 ml 80% FCS, 10%DMSO and 10% RPMI-1640 and stored frozen at −70° C., in 2 ml aliquots.Stored aliquots have been defrosted and retested for viability andantibody specificity.

The hybridoma was deposited on Nov. 14, 1997 with the American TypeCulture Collection (Rockville, Md.) and received ATCC Accession No. ATCCCRL 12431.

EXAMPLE 2

In the Example set forth below, the following Materials and Methods wereused.

LF Proteins

Human milk lactoferrin, obtained from Sigma (L3770) is designated LF(M). Seminal plasma LF was isolated from pooled specimens of semen, fromclinically normal volunteer donors. Following liquefaction, sperm-freeplasma was obtained by centrifugation and separated by DEAE ion exchangechromatography (11) into a pool of basic and a pool of acidic fractions.Each pool was subjected to gel filtration (SEPHACRYL™ S 300 HR,available from Amersham Pharmacia of Piscataway, N.J.), and the firstfraction of each pool was resolved at 80 kD and designated SP80-basicand SP80-acidic, respectively.

Cyanogen Bromide (CNBr) Cleavage and SDS PAGE

CNBr treatment of SP80-basic, SP80-acidic and LF(M) was carried out asdescribed (12). Briefly, a 10 mg/ml 70% formic acid solution of eachprotein was incubated with CNBr (200 fold molar excess) at roomtemperature for 18-24 hr. Following lyophilization, the cleavagemixtures were electrophoresed on an SDS polyacrylamide gel (FIG. 1). Forenhanced resolution of the low molecular weight fractions (FIG. 2)electrophoresis was carried out on a 16.5% tricine gel (13).

Characterization of Fraction 7B

Fraction 7B was excised from the gel and extracted with H20-SDS wasprecipitated by addition of KC1 and the component peptides of 7B werepurified by dialysis against PBS (pH 7.2). Untreated sperm-free seminalplasma proteins and native LF were PBS solutions. Determination that LFfraction 7B consisted of two peptides was carried out by the Laboratoryof Mass Spectrometry at Rockefeller University, utilizingmatrix-associated laser desorption/ionization mass spectrometry (14).N-terminal sequencing of the peptides of LF fraction 7B was carried outby the Protein Sequencing Facility at Rockefeller University, utilizingrepeated cycles of Edman degradation followed by PTH analysis withmicrobore HPLC (15).

Immunoreactivity

Western blot was performed on Immobilon-P (Millipore) transfers of theelectropherograms of LF(M) and acidic and basic SP80 and visualized bychemiluminescence. ELISA was carried out by standardized methodology(16-18). Sera were those of a rabbit immunized with human LF(M), arabbit immunized with SP80 (acidic and basic combined) and human seraselected at random from a group of discards from clinical laboratories,identified by gender, age and “no clinical findings”. Reactivity by allhuman sera was solely with fraction 7 of the PAGE (FIG. 1) and resolvedat a distinct band designated 7B (FIG. 2).

Monoclonal Antibody (Mab) Specific for Fraction 7B

Mononuclear cells were isolated from cord blood of a normal neonate bydensity gradient centrifugation using FICOLL-PAQUE® (Pharmacia) andtransformed with Epstein-Barr virus (19). Fusion with the parental cellline HMMA, utilizing standard procedures (20), resulted in a set of IgMsecreting hybridomas for which monoclonality was established by limitingdilution. Since reactivity of serum with denatured milk LF(M) and SP80was confined to a single PAGE fraction (FIG. 2) that fraction wasisolated from the gel and utilized, together with a set of proteins andpeptides for which specific reactivity by other human natural antibodieshas been established (16,18), as antigens in ELISA to screen those Mab'sfor exclusive reactivity with fraction 7B.

Cytologic Localization of LF/SP 80 in Sperm Heads.

A fraction of swim-up human sperm was obtained from spontaneouslyliquefied seminal plasma, washed 3 times with PBS, and fmally suspendedin either human serum diluted 1:500 in PBS or in PBS solution of thepurified Mab, followed by overnight incubation at 40° C. Each suspensionwas washed 3 times with PBS and the collected sperm incubated in FITClabeled anti-human IgM (Sigma) for 1 hour. The sperm were washed withPBS, and a drop of the suspension placed on a slide, examined andphotographed, utilizing FITC-specific filters (FIG. 3).

Sperm Coat Protein Fraction

A fraction containing the components of the sperm coat was obtained byinduction of the acrosome reaction (21) in a suspension of spermatozoa:the swim-up sperm were gently washed with PBS, collected and suspendedin Ca medium: 2 mM CaCl₂10 mM ionophore A23187 (Calbiochem), 1 mM PMSF(Sigma) and incubated 4 hr at room temperature. The sperm cells werepelleted by low speed centrifugation and the resultant supernatantcleared of particles by high speed centrifugation followed by dialysisovernight at 40° C. The supernatant was tested by ELISA, for reactivitywith human sera and with the Mab reactive with LF fraction 7B (FIG. 4).

Results

The data reported here confirm previous studies indicating that an 80 kDprotein of human seminal plasma is homologous with LF (1,2).Fractionation of sperm-free seminal plasma by DEAE ion exchangechromatography (not shown) confirmed that the 80 kD protein is presentin two forms: basic and acidic, which contains the glycan moiety (22).CNBr cleavage fractions on SDS gels were identical for both forms ofSP80 as well as for LF derived from human milk (FIGS. 1A,2A). Also, thepattern of immunoreactivity of those fractions with serum of a rabbitimmunized with SP80 (FIG. 1) or with LF from human milk (not shown), arecorrespondingly identical. Similarly, prior reports (4) that normalhuman sera show no immunoreactivity with native LF from milk or withSP80 isolated from, or in the context of, seminal plasma are confirmed(FIG. 4). Especially significant is the confirmation (FIGS. 1, 2) that anatural antibody, identified in normal human sera (3, 4), is reactivewith a cryptic sequence of LF and SP80 that is revealed upondenaturation of those proteins (FIGS. 1, 2). That sequence is segregatedin fraction 7B from the PAGE of CNBr cleavage products of LF(M) and SP80(FIG. 2). The innate occurrence of the natural antibody is strikinglydemonstrated by the derivation of a hybridoma from a cord blood cellwhich secretes an IgM/K that is specifically reactive with a componentof fraction 7B (FIGS. 2, 4).

Mass Spectrometry revealed that fraction 7B contains 2 peptides, 10 kDand 9 kD. N-terminal sequencing identified DKVER (amino acid positions1-5 of SEQ ID NO: 13) for the load major peptide and SLDGG (amino acidpositions 1-5 of SEQ ID NO: 27) for the 9 kD peptide. Upon theassumption that CNBr cleavage of LF is at methionine residues and byreference to the published structure of LF (12) the sequence of each ofthe 2 peptides was derived and localized to the C lobe. A set of 12residue peptides, with 5 residue overlaps, comprising the derived linearsequences of the 2 peptides, was created (Table I). Thus far, specificreactivity of human serum IgM has not been identified with any one ofthose peptides tested singly, indicating that the fundamental epitopefor the natural antibody, although embodied in LF fraction 7B, isconformation dependent.

The localization of that epitope, in situ in the sperm head, isdemonstrated by cyto-immunoreactivity of human serum and by the Mabspecifically reactive with LF(M)/SP80 fraction 7B (FIG. 3) Furtherevidence that LF is present in the sperm coat proteins, in thatconfiguration in which the natural antibody epitope is revealed, isprovided by FIG. 4. Following induction of the acrosome reaction (21),resulting in dispersion of the protein coat/plasma membrane ensembleoverlying the acrosomal region of the sperm head, reactivity of acomponent of the coat with human serum IgM and with the Mab was shown(FIG. 4). Thus, FIGS. 3,4 provide evidence that, following the sequenceof capacitation and acrosome reaction, in vivo the LF shed from thesperm coat may be available for entry into the sperm-penetrated oocyte.However, since the complete immunoglobulin repertoire of plasma ispresent in the female reproductive tract (23) that availability may beinhibited by the natural antibody.

Table I. Overlapping duodecapeptides comprising the components of LFfraction 7B. A. 10 kD, B. 9 kD

TABLE I Overlapping duodecapeptides comprising the components of LFfraction 7B. A. 10 kD, B. 9 kD Seq. ID No: A. D K V E R L K Q V L L H 13K Q V L L H Q Q A K F G 14 Q Q A K F G R N G S D C 15 R N G S D C P D KF C L 16 P D K F C L F Q S E T K 17 F Q S E T K N L L F N D 18 N L L F ND N T E C L A 19 N T E C L A R L H G K T 20 R L H G K T T Y E K Y L 21 TY E K Y L G P Q Y V A 22 G P Q Y V A G I T N L K 23 G I T N L K K C S TS P 24 K C S T S P L L E A C E 25 S P L L E A C E F L R K 26 B. S L D GG Y V Y T A C K 27 V Y T A C K C G L V P V 28 C G L V P V L A E N Y K 29L A E N Y K S Q Q S S D 30 S Q Q S S D P D P N C V 31 P D P N C V D R PV E G 32 D R P V E G Y L A V A V 33 Y L A V A V V R R S D T 34 V R R S DT S L T W N S 35 S L T W N S V K G K K S 36

As noted (Results) reactivity of human serum IgM or of the Mab was notdisplayed against any of the peptides, indicating that the epitope isconformational.

References

1. Hekman A, Rumke P. The antigens of human seminal plasma (with specialreference to lactoferrin as a spermatozoa-coating antigen). ProtidesBiol Fluids 16:549-552, 1969.

2. Goodman S A, Ycung L G. Immunological identification of lactoferrinas a shared antigen on radioiodinated sperm surface and inradioiodinated human seminai plasma. J. Reprod Immunol 21:99-108, 1981.

3. Rodman T C, Laurence J, Pruslin F H, Chiorazzi N, Winston R.Naturally occurring antibodies reactive with sperm proteins: apparentdeficiency in AIDS sera. Science 228:1211-1215, 1985.

4. Manchester K, Winston R., Rodman T C. Lactoferrin-reactive naturalantibodies. Ann NY Acad Sci in press.

5. Boyden S V. Natural antibodies and the immune response. Adv Immunol5:1-28, 1965.

6. Guilbert B, Dighiero G, Avrameas S. Naturally occurring antibodiesagainst nine common antigens in human serum. Detection, isolation andcharacterization. J Immunol 128:2779-1787, 1982.

7. He J, Furmanski P. Sequence specificity and transcriptionalactivation in the binding of lactoferrin to DNA. Nature 373:721-724,1995.

8. Bi BY, Liu J L, Legrand D, Roche A-C, Capron M, Spik G, Mazurier J.Internalization of human lactoferrin by the Jurkat human lymphoblastic Tcell line. Eur J Cell Biol 69:288-296, 1996.

9. Yanagimachi R. Mammalian fertilization. In: Knobil E, Neil J D, Eds.The Physiology of Reproduction. New York: Raven Press, pl89-317, 1994.

10. Aitken R J. Fertilization and early embryogenesis. In Hillier S G,Kitchener H C, Neilson J P, Eds. Scientific Essentials of ReproductiveMedicine. London: W. B. Saunders, p2.10, 1996.

11. Friesen A D, Bowman J M, Price H W. Column ion exchange preparationand characterization of an Rh immune globulin for intravenous use. JApplied Biochem. 3:164-175, 1981.

12. Metz-Boutigue M-H, Joll&s J, Mazurier J, Schoentgen F, Legrand D,Spik G, Montreuil J, Joll&s P. Human lactoferrin: amino acid sequenceand structural comparisons with other transferrin. Eur J Biochem145:659-676, 1984.

13. Schagger H, van Jagow G. Tricine-sodium dodecylsulfate-polyacrilamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa. Anal Biochem. 166:368-373,1987.

14. Beavis R C, Chait B T. High accuracy molecular mass determination ofproteins using matrix-assisted desorption mass spectrophotometry. AnalChem 62:1836-1840, 1990.

15. Atherton D, Fernandez J, DeMott M, Andrews L, Mische S M. Routineprotein sequence analysis below ten picomoles. In: Angeletti R H, Ed.Techniques in Protein Chemistry IV, Calif. Academic Press. p409-418,1993.

16. Rodman T C, Pruslin F H, Chauhan Y, To S E, Winston R.Protamine-reactive natural antibodies in human sera. J Exp Med167:1228-1246, 1988.

17. Pruslin F H, To S E, Winston R, Rodman TC. Caveats and suggestionsfor the ELISA. J Immunol Meth 137:27-35, 1991.

18. Rodman T C, To S E, Hashish H, Manchester K. Epitopes for naturalantibodies of human immunodeficiency virus (HIV)-negative andHIV-positive sera are coincident with two key functional sequences ofHIV Tat protein. Proc Natl Acad Sci USA 90:7719-7723, 1993.

19. Chiorazzi N, Wasserman R L, Kunkel HG. Use of Epstein/Barr virustransformed B-cell lines for the generation of immunoglobulin-producinghuman B cell hybridomas. J Exp Med 156:930-935, 1982.

20. Chiorazzi N, Generation of stable autoantibody-secreting B cellhybridomas. Mol Biol Reports 16:65-73, 1992.

21. Jamil K, White I G, Induction of acrosomal reaction in sperm withionophore A23187 and calcium. Arch Androl 7:293-292, 1981.

22. Spik G, Coddeville B, Mazurier J, Bourne Y, Cambillant C, MontreuilJ. Primary and three-dimensional structure of lactotransferrin(lactoferrin) glycans. Adv Exp Med Biol 357:21-32, 1994.

23. Yee A J, Silver L M. Contraceptive vaccine formulations with spermproteins. In: Bronson R A, Alexander N J, Anderson D J, Branch D W.Kutteh W H, eds. Reproductive Immunology. Mass. Blackwell Science. part2, chapt. 33, 1996.

24. Haas G G Jr. Bronson R A, D'Cruz J, Fusi F M. Antisperm antibodiesand infertility In: Bronson R A, Alexander N J, Anderson, D J, Branch DW, Kutteh W H, eds. Reproductive Immunology. Mass. Blackwell Science.part 2, Chapt. 7, 1996.

25. Rodmar, T C, Pruslin F H, To S E, Winston R, Allfrey V G. Turnoverof basic chromosomal, proteins in fertilized eggs: a cytoimmunochemicalstudy of events in vivo. J Cell Biol 90:351-361, 1981.

26. Monchev S,. Tsanev S. Protamine-histone replacement and DNAreplication in the male mouse pronucleus. Mol Reprod Devel 25:72-76,1990.

27. Fleet J C. A new role for lactoferrin: DNA binding and transcriptionactivation. Nutr Rev 53:226-231, 1995.

28. Garre C, Bianchi-Scarra G, Sirito M, Musso M, Ravazzolo R.Lactoferrin binding sites and nuclear localization in K562 (s) cells. JCell Physiol 153:477-482, 1992.

29. Hutchens T W, Henry J F, Yip T T, Hachey D L, Schanler R S, MotiiKJ, Garza C. Origin of intact lactoferrin and its DNA-binding fragmentfound in the urine of milk-fed infants. Evaluation of stable isotopicenrichment. Ped Res 29:243-250, 1991.

30. Concar D, The jaws of lactoferrin. Nature 344:710, 1990.

31. Gerstein M, Andersen B F, Norris G E, Baker E N, Lesk A M, ClothiaC. Two hinges produce a see-saw motion between alternative close-packedinterfaces. J Mol Biol 234:357-372, 1993.

32. Baker E N, Anderson B F, Baker H M, Day C L, Rumball S V, Smith C L,Thomas DH. Three dimensional structure of lactoferrin in variousfunctional states. Adv Exp Med Biol 357:1-12, 1994.

33. Lonnerdal B,Iyer S, Lactoferrin: molecular structure and biologicalfunction. Ann Rev Nutr 15:93-110, 1995.

EXAMPLE 3 Production of RWT-4 and RWT-12 Hybridoma Cell Lines

Each of the hybridomas was prepared by fusion of a human umbilical cordblood B cell with a myeloma.

Myeloma fusion partner for RWT-4 was a heteromyeloma produced by fusionof a mouse and a human myeloma cell, obtained from ATCC: 5HMD33.

Myeloma fusion partner for RWT-12 was HMMA. (Posner, M. R. et al.,Hybridonza 6:611, 1987).

For each hybridoma, umbilical cord blood B cells were obtained andimmortalized as described above in Example 1.

After five weeks, the culture medium of each well was tested by ELISAfor reactivity with Tat protein. The media of selected cultures of thoseEBV-immortalized cells was then tested with each of the peptides (shownin Example 5, FIG. 5). Three of those displaying reactivity only withpeptide 4 (SEQ ID NO: 4) (and at lower levels with peptide 5, SEQ ID NO:5) and three displaying reactivity only with peptide 8 (SEQ ID NO: 8)(and at lower level with peptides 7, 9 (SEQ ID NO: 7, 9)) were selectedfor fusion with the respective fusion partner as described above inExample 1. Note correction to that procedure: the fused cells(representing the hybridomas) were plated out at 0.5 cells per well toassure that no more than one cell was seeded into a well, thus assuringmonoclonality.

The cells were grown in NY/HT (Sigma), supplemented with 20% fetal calfserum, SPIT (Sigma) and Pen/Strep, to a density of 106 cells/ml, andspun at 400 RPM, 5 min.

Each pellet was suspended in medium, consisting of 80% fetal calf serum,10% DMSO and 10% RPMI-1640, and stored, in 2 ml aliquots, at 70° C. orin liquid nitrogen. Those aliquots represent the hybridomas RWT-4 andRWT-12 deposited with ATCC on Feb. 12, 1998 and FEb. 25, 1998,respectively.

EXAMPLE 4 Testing of Specific Reactivity of Hybridoma Cell Lines RWT-4and RWT-12

The culture medium of each, containing the specific IgM monoclonalantibody secreted by the hybridoma, was treated for recovery of theantibody.

The medium was concentrated in a CENTRICON® (available from Millipore ofBedford, Mass.) column to remove salt and all proteins of mol. wt. lessthan 100 Kd. The concentrated solution was then passed through a sizeexclusion gel on a Pharmacia S-300 column. The first peak of eluate wasrun on SDS polyacrylamide gel to inspect purity, demonstrated by displayof two bands, representing the light and the heavy chain of the specificIgM, and no other bands. The eluate was then reconcentrated in a newcolumn to 200 μg/ml.

The light chain of each Mab was identified by ELISA with peroxidaselabelled anti-gamma and anti-kappa.

The heavy chain (epitope specificity) of each was identified by EIISAwith the set of peptides.

LIGHT CHAIN IDENTIFICATION ELISA 1. unlabeled rb > IgM 2. Mab or totalhu IgM (Sigma) 3. perox. labeled anti-kappa or anti-lamda 0 ab Total IgMRWT-4 Mab Perox Labelled Anti-Lambda 1:4k 01 .24 .73 1:6k 01 .16 .541:10k  0 .12 .37 Anti-Kappa 1:4k 04 .55 .06 1:6k 01 .36 .04 1:10 k 0 .23.02 Conclusion: light chain for RWT-4 is lamda 0 ab Total IgM RWT-12 MabAnti-Lambda 1:4k 02 .89 .03 1:6k 01 .65 .02 1:10k  07 .45 .01 Anti-Kappa1:4k 04 >1.00 .80 1:6k 03 .83 .57 1:10k  02 .56 .39 Conclusion: lightchain for RWT-12 is kappa

EPITOPE DETERMINATION IN TERMS OF TAT PEPTIDE SPECIFICITY

ST RWT-4 Mab RWT-12 Mab Pep 1 .01 .03 .05 2 .02 .02 .04 3 .03 .01 0 4.48 .94 .02 5 .20 .35 .12 6 .07 .01 .07 7 .16 .07 .36 8 .12 .07 .42 9 0.02 .01 10 0 .01 .03 11 0 0 0 12 .33 .16 .72 Total Tat Protein .49 .55.44

Peptide 12 (here) is designated peptide 8 in Example 5, FIG. 6.Therefore,

here FIG. 2 7 7 12 8 8 9 9 10 10 11 11 12 12 8

Dilutions: ST serum 1:100

Mab's 1 μl/ml of the hybridoma cells

These data represent the average, for each antibody/antigen reaction, of20 separately run assays.

EXAMPLE 5

In the present Example, the following materials and methods were used.

Sera

Human

The 70 HIV+ and 70 HIV− sera reported in FIG. 6 were collected prior to1994, and assayed for reactivity with Tat protein. Therefore thecharacteristics of the HIV+ cohort are not attributable to the anti-HIVmedications in use since that time. Of those 70 HIV+ sera, 52 wereavailable for the epitope analysis of Table I in which were included 8additional HIV+ sera for a total of 60 sera from HIV+ individuals not onmedication. The sera for the HIV+ serial sets (FIGS. 9, 10, 11) werealiquots of specimens submitted for clinical examination with clinicaldata and concurrent medication noted. The 80 normal (HIV−) sera of TableI were assembled from specimens submitted for pre-employment examinationidentified only by age, gender and “no clinical findings”, and fromdonations by laboratory personnel.

Chimpanzees

A total of 22 sera from adult chimpanzees, certified as normal, wereobtained: 16 (7 ♂, 9 ♀) from YERKES Regional Primate Center (EmoryUniversity); 6 (2 ♂, 4 ♀) from LEMSIP (NYU Medical Center). Serum of 1 ♂and 1 ♀ of the latter group were collected 22 months and 10 months,respectively, post innoculation with HIV infected cells.

Monkeys

A total of 32 sera from normal monkeys were obtained: 20 rhesus macaquesfrom YERKES, 1 from LEMSIP and 2 from LARC (Laboratory Animal ResearchCenter, Rockefeller University), 4 pig tail macaques and 5 baboons fromLARC. Also, serum was obtained from 1 of the rhesus macaques followinginnoculation with SIV (Mac 239) infected cells and 2 specimens of rhesusplasma, 6 months post-innoculation with cell free supernatant of SIV Mac239 culture, were obtained from Dr. Lingi Zhong (Aaron Diamond AIDSResearch Center, Rockefeller University).

Rabbits

Sera obtained from 20 (10 ♂, 10 ♀) New Zealand white rabbits (prior toany treatment) were generously provided by James Nolan (Hospital forSpecial Surgery, New York) and 10 were obtained from LARC. 1 specimen ofrabbit serum post-immunization with HIV Tat protein was obtained fromIntracel Corp (Isaquah, Wash.)

Mice

Sera from 30 normal adult mice: 12 Balb C, 6 C57 black, 2 MRL-lpr, and10 Swiss Webster were obtained through LARC. A series of 3 immunizationswith HIV Tat protein/adjuvant was administered to 1 Balb C and 1 SwissWebster and adjuvant alone was administered to 1 Balb C and 1 SwissWebster. Sera included in the data of Table I represent the specimenscollected 16 weeks after the final innoculation of each mouse.

Antigens

Recombinant Tat protein was obtained from Intracel Corp. in lyophylizedform. Reactivity and working dilution for each vial of the protein wasstandardized with a single (standard) human serum (16). Tat peptides(SEQ ID NOS: 1 to 12) (FIG. 5), representing overlapping sequences inaccordance with the published amino acid alignment of HIV Tat (17) wereprepared as previously described (14). The most recent review (26)confirms that Tat is a highly conserved HIV protein with littledigression from that sequence displayed by the various HIV clades.

Elisa

All sera were stored at −70° C. in small aliquots, to minimize theeffects of repeated freeze-thaw. The ELISA protocol has been rigidlystandardized and statistically evaluated (e.g. 15,16). Eachserum/antigen was tested in a minimum of 3 separate assays. Thecorrected serum O.D. for each antigen represented the read-out O.D. ofthe serum/antigen minus the O.D. of serum background (0 antigen).Corrected O.D. of 0.10 was considered positive. If corrected O.D. was0.08-0.15, the assay was repeated 3 additional times. For assay of humanand chimp sera, a single standard serum (ST) was included on each titerplate and the fmal titer was calculated as X/ST. Peroxidase labeledanti-human IgG or IgM (KPL) was used for all human and chimpanzee sera.Anti-monkey IgM or IgG (KPL) was found to be non-reactive withchimpanzee sera, but was appropriate by all criteria of specificity andserum-dilution proportionality with the different monkey sera tested.Similarly, the anti-mouse IgM or IgG (Sigma) and anti-rabbit IgM or IgG(KPL) were screened for specificity and dilution related gradient ofreactivity. Since the peroxidase labeled antibodies for each specieswere derived from goat serum, the ELISA included an extra blocking step,i.e. 1% normal goat serum applied following the antigen wash and priorto application of the species-specific test serum, to assure that nopart of the displayed reactivity was attributable to goat antibodies.

TABLE 1 IgM IgG Species # of Sera Peptide: 1 4 8 Tat 1 4 8 Tat HumansMales 40 0 40 38 40 0 38 31 38 Females 40 0 40 40 40 1 40 36 40 HIV + 600 60 46 60 1 60 21 60 Chimps Males 11 0 11 10 11 0 11 9 11 Females 11 011 11 11 0 11 8 11 HIV + 2 0 2 2 2 0 2 2 2 Simmians Monkeys 32 0 0 0 0 032 2 21 SIV + 3 0 0 0 1 0 3 3 3 Rabbits Normal 30 0 2 0 0 0 3 0 0 Tat +1 1 0 0 1 1 1 0 1 Mice Normal 30 0 0 0 0 0 2 0 1 Adj. Only 2 0 0 0 0 0 00 0 Tat + 2 0 0 0 0 2 0 0 2

Results

Human

FIG. 6 presents the assay data of IgM and IgG reactivity with Tatprotein of HIV+ and HIV−(normal) sera. As noted in METHODS, those HIV+sera were collected from individuals who had not received any anti-HIVmedication other than that in general use prior to mid-1994 (e.g. AZT).Comparison of the assembly of titers of the two cohorts of 70 sera each,shows that the IgM titers (FIG. 6A) of the HIV+ cohort are atsignificantly lower levels than those of the HIV− cohort. Thedistribution of the Tat-reactive IgG titers of the same sera (FIG. 6B),however, appears to be random, both with respect to comparison of thetwo cohorts and, in individual sera (not shown), in relation to theTat-reactive IgM titers. Those IgG titers may represent maturation formsof the natural antibodies (27,28) or antibodies independently induced byunrelated antigens with sequences sufficiently concordant with regionsof Tat protein to be reflected as Tat-reactive.

Epitope analysis (Table I) of sera of each of the two human cohortsshows that the entire IgM reactivity with Tat protein is limited to twonon-adjacent sequences: one including peptides 4,5 embracing thecysteine-rich region and the other peptides 7,8,9 representing thearginine rich region (FIG. 1). In accord with the data of Table I, all(80) HIV− (normal) males and females have significant titers of IgMreactive with Tat protein as well as with the epitope represented bypeptide 4 (SEQ ID NO: 4), while all but 2 have significant titers withthat represented by peptide 8 (SEQ ID NO: 8). All of the 60 HIV+ serahave low, but significant, titers of IgM antibodies reactive with Tatprotein and the sequence represented by peptide 4 (SEQ ID NO: 4), whileonly 46 of the 60 have IgM reactive with the arginine-rich sequencerepresented by peptide 8 (SEQ ID NO: 8). For even those HIV+ sera thatare within the range of positive, the IgM reactivities with peptide 8(SEQ ID NO: 8) are at low levels (FIG. 7), clearly suggestive of a trendto depletion, more so than that of peptide 4 (SEQ ID NO: 4) (FIG. 8).Again, the IgG antibodies (Table I) may be considered to representmaturation forms (27, 28) of the IgM natural antibodies and/or thoseindependently induced by some exogenous antigenic factor. The latter isprobably applicable to the IgG reactive with peptide 1 (SEQ ID NO: 1)(FIG. 5), present in one HIV-serum, therefore not Tat induced, and oneHIV+serum (Table I). The data of FIGS. 7 and 8 confirm that the declineof the Tat reactive natural antibodies is more stringently reflected inthat reactive with peptide 8 (SEQ ID NO: 8) (FIG. 7) than in thatreactive with peptide 4 (SEQ ID NO: 4) (FIG. 8). The correlation of thetiters of Tat reactive IgM natural antibodies with the pathoprogressionof HIV and with the CD4+T cell count, an established index of thatprogression (29), is shown in FIGS. 9, 10, 11. Each is a display of dataobtained from serial specimens of a single individual, including IgMassay titers for Tat protein, peptide 4 (SEQ ID NO: 4), peptide 8 (SEQID NO: 8) and clinical laboratory report of CD4+T cell counts. Theseries in FIG. 9 is that from an HIV+ male collected over a period offive years preceding his death with a diagnosis of AIDS. Each value forTat protein IgM titer reflects the combination of the peptide 4 (SEQ IDNO: 4) and peptide 8 (SEQ ID NO: 8) IgM values for the same specimen.Particularly striking is the sharp rise followed by the precipitous dropin the peptide 8 (SEQ ID NO: 8) reactivity concurrent with the virtualwipe-out of the CD4+T cells in the specimen collected 8 months prior todeath. FIG. 10 is a display of data of the series of specimens from anHIV+ male whose duration of infection is estimated at over 11 years andwho has had no anti-HIV medication and no symptoms of HIV pathogenesisand, thus, fits the criteria of long-term -survivor (LTS) orlong-term-non-progressor (LTNP) (12,13). The pattern of maintenance oftiters of the IgM natural antibodies reactive with Tat protein, peptide4 (SEQ ID NO: 4) and peptide 8 (SEQ ID NO: 8) are similar to thosedefmed for normal (HIV−) humans (14,16). The high levels of titers,particularly those for peptide 8 (SEQ ID NO: 8), are correlative withthe maintenance of CD4+T cell counts within the normal range. Similarcorrelation is shown in the series of specimens (FIG. 7) from a singleHIV+ individual for whom antiviral therapy was initiated followingreport of decline in CD4+T cell count. Following a period of medication,both CD4+T cell count and the titers of the natural antibodies,particularly those reactive with peptide 8 (SEQ ID NO: 8), rose. Thefollowing successive specimens showed maintenance of both CD4+T cellcounts and antibody titers in the normal range, concomitant with ageneral state of wellness of the patient.

Chimpanzee

The sera of all of the 22 normal chimps (Table I) had significant titersof both IgM and IgG antibodies reactive with Tat protein and peptide 4(SEQ ID NO: 4). For peptide 8 (SEQ ID NO: 8), 21 of that group displayedsignificant IgM and 17 displayed significant IgG reactivity. The sera ofeach of the 2 HIV innoculated chimps displayed significant IgM and IgGreactivity with Tat protein, with the sequences represented by peptides4 (SEQ ID NO: 4) and 8, (SEQ ID NO: 8) and with no other. Thus, thenatural antibody repertoire of chimpanzee is similar to that of humans.

Monkey

No IgM reactive with Tat protein or any of its constituent peptides wasdetected in the sera of any of the 32 normal sera (Table I). Of the 3SIV infected monkeys, one showed reactivity with Tat protein. All 32,however, displayed IgG reactivity with peptide 4 (SEQ ID NO: 4) and 21of those displayed IgG reactivity with Tat protein. Two sera of thenormal macaques and all three of the SIV infected macaques displayed IgGreactivity with peptide 8 (SEQ ID NO: 8).

Rabbit

Of the 30 normal rabbit sera, two displayed IgM reactivity with peptide4 (SEQ ID NO: 4) which, however, was not accompanied by detectable IgMreactivity with Tat protein. Those two and an additional normal rabbitserum displayed IgG reactivity with peptide 4 (SEQ ID NO: 4) but, again,not with Tat protein. The Tat immunized rabbit serum displayed IgMreactivity with peptide 1 (SEQ ID NO: 1) and with Tat protein and IgGreactivity with peptide 4 (SEQ ID NO: 4) as well as peptide 1 (SEQ IDNO: 1) and Tat protein. That distribution suggests that the peptide 4(SEQ ID NO: 4) IgM and IgG reactivity in both normal and Tat immunizedrabbit serum reflects a response to an exogenous antigen that is notdetectable in the assembled Tat protein. The IgM and IgG reactivity withpeptide 1 (SEQ ID NO 1), displayed in the serum of the Tat immunizedrabbit is attributable to induction by the immunogen since that peptide1 (SEQ ID NO: 1) reactivity is reflected in comparably high reactivitywith Tat protein.

Mouse

Of the sera from 30 normal, two Tat/adjuvant and two adjuvant/onlyimmunized mice, none displayed IgM reactivity with Tat protein or any ofthe peptides (Table I). Two of the 30 normal mouse sera displayed IgGreactivity with peptide 4 (SEQ ID NO: 4) and the serum of another mousedisplayed IgG reactivity with Tat protein. The sera of the two miceimmunized with adjuvant/only displayed no reactivity while the sera ofthe two mice immunized with Tat/adjuvant displayed exceedingly high(>1.0) activity with peptide 1 (SEQ ID NO: 1) and with Tat protein.Clearly, for both rabbit and mouse, Tat protein is a potent inducer ofan antibody response specifically directed to the sequence displayed inpeptide 1 (SEQ ID NO: 1).

Discussion

The significance of the Tat protein is shown early in the pathogeneticsequence of HIV infection by its role in cell attachment and entry ofthe virus. Evidence from in vitro study indicates that Tat participatesin viral internalization, mediated primarily by the basic domain(30,31), represented by Tat peptides 7, 8, 9 (SEQ ID NOS: 7, 8, 9) (FIG.5). Intracellular propagation of the virus is also dependent upon Tatthrough its interaction with the Tar region of the viral RNA, resultingin transactivation (18, 19). The cysteine region of Tat, represented byTat peptides 4,5 (SEQ ID NOS 4, 5) (FIG. 5) plays an essential role inTat/Tar binding and the consequent replication of HIV (18,19). Thus, twoactivities of Tat—mediation of viral cell entry and activation of theinternalized virus to replicate—are dependent upon the sequences of Tatthat include the epitopes for the two natural IgM antibodies that arepresent in the sera of all human and chimpanzee sera examined in thisstudy, but are not present in the sera of other mammals, e.g. monkeys,rabbits, mice (Table I)

In accord with that epitopic specificity, we propose that those naturalantibodies provide, or contribute to, the human host mechanism ofresistance to HIV pathogenesis in the early post-HIV infection period.Retardation of viral entry and intracellular replication by thoseantibodies in the human host and absence of that retardation in rhesusmacaques may account for the observations that T lymphocyte turnover inSIV infected rhesus macaques occurs at a considerably higher rate thanthat in HIV infected humans (32,33). Although the precise mechanismswhereby the CD4+T cell population is depleted in the peripheral bloodcells of HIV+ humans are not yet specifically established, arelationship between the CD4+T cell count and titers of the Tat-reactivenatural antibodies is demonstrated in the serial specimens of FIGS. 9,10, 11 of this study. In each series, the CD4+T cell counts parallel themaintenance and drop of the antibody titers.

However, the providential arrest of Tat-related pathogenicity by thosenatural antibodies may be limited by the immune system recognition ofthe antibody-reactive sequences of Tat as self antigens and theconsequent induction of tolerance (21, 22).

The separate and coordinate principles of innate and adaptive immunityhave received much attention recently (6,7,8) which, hopefully, willprovide further elucidation of the mechanisms and events of selfrecognition followed by tolerance. Thus far, the fundamental andimplemental event of self tolerance appears to be that of deletion, orturning off, of the T and/or B cells involved in natural antibodyproduction (23). Thus, as the Tat antigen load is increased, theproduction of Tat-reactive natural antibodies may be stifled,antibody-mediated restriction of the aggressive activities of Tat lost,and the period of pathoprogressive latency terminated. A pathogenicactivity of Tat, well documented in vitro, is that of induction ofapoptosis (29). The proposition that the Tat-reactive natural antibodiesmay impede the action of Tat, and thereby contribute to maintenance ofthe early period of apparent latency following HIV infection, issupported by the observation that persons designated LTS (long termsurvivor) (12) or LTNP (long term non progressor) (13) show littleevidence of T cell apoptosis (21) and, as we have shown (FIG. 10),maintain normal levels of the natural antibodies. In correlation arereports (34) that the resistance of chimpanzees to progress to AIDS isaccompanied by maintenance of T cell levels and little evidence of Tatinduced apoptosis.

Although the mechanisms underlying depletion of T cells by apoptosis arenot completely understood, recent studies have established that theFas/Fas ligand system is not the modulating factor in HIV inducedapoptosis of CD4+T cells (35, 36). Particularly provocative, however, isa recent report that SIV (mac 239) induced apoptosis in peripheral bloodmononuclear cells in vitro is mediated by the Fas/Fas ligand system(37). That difference between SIV and HIV in the mechanism of apoptosismediation is critically relevant to the thesis of this study—that theinteraction of HIV with the human immune system is significantly unique.Another significant difference between HIV and SIV is indicated by invitro studies of the effect of intervention by interferon on viralreplication. That effect appears to be primarily concerned with viralDNA synthesis which, in SIV infected cells, is blocked by interferonbut, in HIV infected cells, is not (38).

Apoptosis of B cells as well as T cells has been attributed to action byTat (29). Even more compelling are the accumulating reports of theinvolvement of Tat in the neurodegeneration leading to dementia (24).The reports have included Tat dose-dependent apoptosis of human fetalneurons in culture (39) and neuronal apoptosis detected in brain tissuefrom patients who had died with a diagnosis of AIDS (40). Theprobability that the neurotoxic effects of Tat demonstrated in vitro mayoccur in vivo is supported by the potential ability of Tat to permeatethe blood brain barrier. Various analyses of the capacity for vascularpermeability and blood brain barrier passage by the sperm chromosomalprotein, protamine, have assigned that function to the arginineconcentration of protamine (41). That same capacity is inherent in thearginine rich sequence of Tat, represented here by peptides 7,8,9 (SEQID NOS: 7, 8, 9) (FIG. 5). Of particular relevance is the epitopeanalysis for the human natural antibody reactive with Tat peptide 8 (SEQID NO: 8) which showed (16) that the epitope for that natural antibodyis present in certain arginine-rich sequences of protamine as well as inHIV Tat protein.

The epitope similarity for the IgM and IgG for each of the two humannatural antibodies suggests that each represents a pair of isotype ofthe same antibody. We have previously proposed that the constancy of IgMtiters, but not the IgG titers, of the two natural antibodies in serialspecimens, from each of a group of normal individuals, indicates thatthe IgM is the homeostasis- maintaining isotype (16). The mechanism andutility of class switch of natural antibodies are currently not wellunderstood nor readily apparent but, hopefully, will be clarified in thecourse of current investigations of the molecular and functional aspectsof the switch of isotype in innate as well as adaptive immunity (42).

Therefore, at present, assignment of separate roles to the IgM and IgGisotope of the human Tat reactive natural antibodies is not feasible.However, it is clear that, in HIV+ humans, Tat-reactive antibodiesattributable to immunogenic induction do not occur (Table I). SinceTat-reactive antibodies are induced in monkeys, rabbits and mice (TableI), it appears that the failure is unique to the human immune system.Since chimpanzees are presumed to have high level of genetic identitywith humans (20), attribution of that uniqueness to genetic specificityis supported by the profile of Tat reactive antibodies in the chimpanzeesera (Table I). The parallel with human sera is evident: pre and postHIV infected chimps have IgG and IgM antibodies reactive with Tatpeptides 4 and 8 (SEQ ID NOS: 4 and 8) (FIG. 5) and with no other (TableI). However, the apparently greater innate resistance of chimps to thepathoprogression to AIDS than that of HIV+ humans (10, 11) may be adeparture from the genetic identity, possibly in some immune systemcomponent participating in induction of tolerance (21, 22). The questionthen arises: is the same genetic characteristic related to theprotection against the ravages of HIV with which LTS/LTNP are endowed(12, 13)?

References for Example 5

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36 1 12 PRT Human 1 Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys 1 510 2 12 PRT Human 2 Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro Lys 1 510 3 12 PRT Human 3 Gly Ser Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr 1 510 4 12 PRT Human 4 Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His 1 510 5 12 PRT Human 5 Lys Cys Cys Phe His Cys Gln Val Cys Phe Ile Thr 1 510 6 12 PRT Human 6 Val Cys Phe Ile Thr Cys Ala Leu Gly Ile Ser Tyr 1 510 7 12 PRT Human 7 Leu Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln 1 510 8 12 PRT Human 8 Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro 1 510 9 12 PRT Human 9 Lys Lys Arg Arg Gln Arg Pro Arg Arg Pro Gln Gly 1 510 10 12 PRT Human 10 Arg Pro Pro Gln Gly Ser Gln Thr His Gln Val Ser 15 10 11 12 PRT Human 11 Thr His Gln Val Ser Leu Ser Lys Gln Pro Thr Ser1 5 10 12 12 PRT Human 12 Lys Gln Pro Thr Ser Gln Arg Gly Asp Pro ThrGlu 1 5 10 13 12 PRT Human 13 Asp Lys Val Glu Arg Leu Lys Gln Val LeuLeu His 1 5 10 14 12 PRT Human 14 Lys Gln Val Leu Leu His Gln Gln AlaLys Phe Gly 1 5 10 15 12 PRT Human 15 Gln Gln Ala Lys Phe Gly Arg AsnGly Ser Asp Cys 1 5 10 16 12 PRT Human 16 Arg Asn Gly Ser Asp Cys ProAsp Lys Phe Cys Leu 1 5 10 17 12 PRT Human 17 Pro Asp Lys Phe Cys LeuPhe Gln Ser Glu Thr Lys 1 5 10 18 12 PRT Human 18 Phe Gln Ser Glu ThrLys Asn Leu Leu Phe Asn Asp 1 5 10 19 12 PRT Human 19 Asn Leu Leu PheAsn Asp Asn Thr Glu Cys Leu Ala 1 5 10 20 12 PRT Human 20 Asn Thr GluCys Leu Ala Arg Leu His Gly Lys Thr 1 5 10 21 12 PRT Human 21 Arg LeuHis Gly Lys Thr Thr Tyr Glu Lys Tyr Leu 1 5 10 22 12 PRT Human 22 ThrTyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala 1 5 10 23 12 PRT Human 23Gly Pro Gln Tyr Val Ala Gly Ile Thr Asn Leu Lys 1 5 10 24 12 PRT Human24 Gly Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro 1 5 10 25 12 PRTHuman 25 Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys Glu 1 5 10 26 12PRT Human 26 Ser Pro Leu Leu Glu Ala Cys Glu Phe Leu Arg Lys 1 5 10 2712 PRT Human 27 Ser Leu Asp Gly Gly Tyr Val Tyr Thr Ala Cys Lys 1 5 1028 12 PRT Human 28 Val Tyr Thr Ala Cys Lys Cys Gly Leu Val Pro Val 1 510 29 12 PRT Human 29 Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Lys 15 10 30 12 PRT Human 30 Leu Ala Glu Asn Tyr Lys Ser Gln Gln Ser Ser Asp1 5 10 31 12 PRT Human 31 Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn CysVal 1 5 10 32 12 PRT Human 32 Pro Asp Pro Asn Cys Val Asp Arg Pro ValGlu Gly 1 5 10 33 12 PRT Human 33 Asp Arg Pro Val Glu Gly Tyr Leu AlaVal Ala Val 1 5 10 34 12 PRT Human 34 Tyr Leu Ala Val Ala Val Val ArgArg Ser Asp Thr 1 5 10 35 12 PRT Human 35 Val Arg Arg Ser Asp Thr SerLeu Thr Trp Asn Ser 1 5 10 36 12 PRT Human 36 Ser Leu Thr Trp Asn SerVal Lys Gly Lys Lys Ser 1 5 10

What is claimed is:
 1. A hybridoma cell line having Accession No. ATCCCRL
 12477. 2. An isolated human IgM monoclonal antibody produced by thehybridoma of claim 1.